Studies On The Molecular Modification Mechanism Of Chitosan Derivatives And Its Application In Preserving Of Silver Pmfret

Aquatic product(such as silvery pomfrets)has been widely accepted by the consumers due to its high protein,low fat,especially low cholesterol.However,the fish meat is highly perishable after postmortem aging.With the fast development of living standards in China,the demands of high quality aquatic foods are kept increasing.On the other hand,traditional low temperature storage could not completely meet the needs.Therefore,to extend the shelf life and maintain the nutritional value of the fish meat remains one of the key scientific problems to be solved in the field of aquatic foods.Natural food biopolymer-based edible packaging material is one of the effective ways applied.Chitosan(CS)is a naturally biopolymer that has good biocompatibility and biodegradability,however,the poor antioxidative and antimicrobial activities of pure chitosan films have limited their application.Thus,green chitosan derivatives(chitosan-g-gallic)were obtained by grafting of gallic acid(GA)to chitosan in this study,and the preservation effect of chitosan-g-gallic acid coating on quality maintenance,spoilage microorganisms,volatile compound and protein denaturation and degradation of refrigerated silver pomfret(Pampus argentus)were investigated.The preservation mechanism was also discussed.The main results are as follows:(1)The chitosan derivatives(chitosan-g-gallic acid)were obtained by H2O2/Vc free-radical-initiated grafting of gallic acid(GA)onto chitosan(CS)in this work.The chemical structures of the chitosan-g-gallic acid were corroborated by UV-vis,GPC,NMR,XRD and DSC analysis.The results showed that gallic acid was most likely interacted with the active hydrogen of NH2 at C-2(amide linkages)and the OH groups at C-6 position(ester linkages)of the chitosan chain.The antioxidant activity(the DPPH radical scavenging activity and reducing power)and antimicrobial activities were enhanced in comparison to pure chitosan.These results showed chitosan-g-gallic acid derivatives as a potential novel biopreservation.(2)The chitosan derivatives(chitosan-g-gallic acid)with different gallic acid degree of substitution(DS)and their corresponding active packaging were prepared.The effect of the DS on the flow and viscoelastic properties of chitosan-g-gallic acid film-forming solutions(FFS),the mechanical,water vapor barrier(WVP),water solubility,contact angle and antioxidant and antioxidant activities and the microstructures,as well as the relationship among these properties of the resulting films,were also discussed.The result showed that the shear-thinning flow behavior of chitosan-g-gallic acid film-forming solutions decreased with the grafting amount of gallic acid into chitosan chain,while the chitosan-g-gallic acid film-forming solutions grafted at a lower gallic acid value behaved like an entangled network system with a more elastic behavior.The increasing of gallic acid grafting onto the chitosan chain led to a reduction of tensile strength,elongation at break and water resistance in the corresponding films,but increases in the antioxidant and antimicrobial activities were observed.The microstructure of the film was investigated using scanning electron microscopy and was closely related to the observed film properties.Upon introducing gallic acid into the chitosan chain,the-OH group of gallic acid could form more intra-and extra-molecular hydrogen bonds and physical junction among CG chains,leading to the changes of the rheological properties of chitosan-g-gallic acid film-forming solutions and structure as well as properties of corresponding film.(3)The effect of chitosan-g-gallic acid conjugates(chitosan-g-gallic acid,CG)coating on the quality of silvery pomfret(Pampus argenteus)during refrigerated storage at 4℃ for 15 days was investigated.The total mesophilic and psychrotrophic microbes in control and coated fish samples were counted periodically,physico-chemical analyses to determine pH,total volatile basic nitrogen(TVB-N),peroxide value(POV),thiobarbituric acid-reactive substances(TBARS),K-value and water-holding capacity(WHC)were carried out,and sensory attributes were evaluated.The results indicated that both chitosan+gallic acid and CG coatings effectively preserved the silvery pomfret quality during refrigerated storage.However,CG had not only higher antimicrobial activity but also a better ability to inhibit the formation of volatile bases(TVB-N)and lipid oxidation products(POV and TBARS)as well as the degradation of ATP and its breakdown products(K value)than that of chitosan+ gallic acid during the storage period.Furthermore,the CG coating also helped to maintain a higher WHC and had better sensory properties for a longer period of time compared to the other treated samples,thus extending the shelf life and delaying the deterioration of fresh silvery pomfret by 3-6 d during refrigerated storage.These results suggested that a CG coating could be employed as an active packaging material in the fish preservation field.(4)The changes of volatile compounds of silvery pomfrets during refrigerated storage were analysed by HS-SPME-GC/MS.The influence of chitosan derivatives(chitosan-g-gallic acid,CG)coating on volatile compounds of silvery pomfrets were also investigated.The aldehydes,esters and alcohol content of silvery pomfrets meat were declined during storage,but ketone,aromatichitosan and amine compounds were showed opposite trend.The changes of volatile compounds(aldehydes,esters,alcohol,ketone,aromatichitosan)of silvery pomfrets were decreased by CG coating.Meanwhile,the formation of undesirable volatile compounds(such as p-xylene,trimethylamine and dimethylamine)was inhibited.These results suggested CG coating could retrain the lipid oxidation of fish meat and thus influence the changes of volatile compounds of silvery pomfrets during refrigerated storage.(5)The dynamic changes of microflora of silvery pomfrets during refrigerated storage were analysed by PCR-DGGE technology.The influence of chitosan-g-gallic acid(CG)coating on these changes was also discussed.The result showed that Pseudomonas sp.,Acinetobacter sp.and Morganella is the main microflora of silvery pomfrets during refrigerated storage.Except these three microorganism,Pseudomonas putida,Psychrobacter fluorescens,Flavobacterium,Psychrobacter glacincola,Providencia,Brochothrix thermosphacta,Shewanella putrefaciens and Shewanella baltica were observed in the fish meat at the end of storage.CG coating could effectively reduce the species and amount of microorganism in the fish meat.The main microflora of CG coating silvery pomfrets is Pseudomonas sp.,Pseudomonas putida,Acinetobacter sp.and Psychrobacter glacincola,indicating CG coating could delay spoilage of silvery pomfrets during refrigerated storage.(6)To explore the influence of CG on the changes of protein properties of silvery pomfrets during refrigerated storage,silvery pomfrets protein denaturation and degradation during storage were investigated in this part.The result showed that CG coating could delay the exposure of the hydrophobic group,the oxidation of sulfhydryl group,reducing the degree of protein denaturation.Meanwhile,17 differential protein spots of CG coating group were identified by 2-DE technology.These differential protein spots were further analysed by MALDI-TOF/TOF technology,and five protein spots were successfully identified by Mascot search,including myosin heavy chain,partial[Cyprinus carpio],actin,alpha skeletal muscle,phosphoglucomutase-1[Ictalurus punctatus],creatine kinase M-type[Fundulus heteroclitus]and phosphoglycerate kinase 1[Takifugu rubripes].Additionally,combined with the change trends of these proteins during storage,suggesting CG coating might delay the degradation of fish protein at a certain extent,and thus prolong its shelf life.